Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
  • C12P7/58—Aldonic, ketoaldonic or saccharic acids
  • C12P7/60—2-Ketogulonic acid
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S435/00—Chemistry: molecular biology and microbiology
  • Y10S435/8215—Microorganisms
  • Y10S435/822—Microorganisms using bacteria or actinomycetales
  • Y10S435/874—Pseudomonas

Definitions

• the present invention relates to a method for producing 2-keto-L-gulonic acid which is useful as a precursor for the synthesis of vitamin C (L-ascorbic acid) and a novel microorganism belonging to the genus Pseudomonas to be used in said production method.
• the method for production of 2-keto-L-gulonic acid using L-sorbose as a starting material has been known.
• the product can be produced only by an oxidation step without involving any reduction step.
• the methods using bacteria belonging to the genera Gluconobacter, Pseudomonas, Serratia, Achromobacter and Alcaligenes have been heretofore known [see Biotechnology and Bioengineering] 14, 799 (1972), Acta Microbiological Sinica, 20, 246 (1980) and 21, 185 (1981), Japanese Patent Publication No. 41-159 and No. 41-160, U.S. Pat. No. 3,043,749, Japanese Patent Publication No. 49-39838 and U.S.S.R. Patent No. 526,660.
• the methods for production of 2-keto-L-gulonic acid from L-sorbose by strains which have been disclosed gives extremely low product yield, and they can not be industrially utilized.
• the present inventors In order to obtain a strain which can produce 2-keto-L-gulonic acid from L-sorbose in a high yield, the present inventors have isolated various strains from soil samples collected in Japan and screened them. As the result, three strains which give extremely higher yield than the earlier results (about 80% based on the sugar consumed) i.e. isolated No. 526-21, No. 526-22 and No. 526-42 have been found. The present inventors have studied these three strains intensively, and found that they belong to a novel species of the genus Pseudomonas. Thus, the present invention is attained.
• the present invention provides a method for producing 2-keto-L-gulonic acid which comprises contacting L-sorbose with a microorganism of Pseudomonas sorbosoxidans having an ability to oxidize L-sorbose to 2-keto-L-gulonic acid or a processed material thereof to produce and accumulate 2-keto-L-gulonic acid and harvesting it.
• the present invention provides a novel bacterial species, Pseudomonas sorbosoxidans which is motile rod bacterium having two or more polar flagella, and which produces no hydroxyacetone from glycerol, contains ubiquinone having 10 isoprene units, and requires thiamine, riboflavine and pantothenic acid for its growth.
• Pseudomonas sorbosoxidans which is motile rod bacterium having two or more polar flagella, and which produces no hydroxyacetone from glycerol, contains ubiquinone having 10 isoprene units, and requires thiamine, riboflavine and pantothenic acid for its growth.
• the three strains which the present inventors have found have the following taxonomic characteristics.
• Nitrate reduction weakly positive.
• the strain can grow at 15° to 36° C.
• the optimum; growth temperature is about 30° C.
• the strain can grow at pH 5.5 to 8.7.
• the optimal growth pH is 6.0 to 7.5.
• Acid is slightly produced but gas is not produced from L-arabinose, D-xylose, D-glucose, D-mannose, D-fructose, D-galactose, maltose, sucrose, lactose, and trehalose. Neither acid nor gas is produced from D-sorbitol, D-mannitol, inositol, glycerol, and starch.
• Thiamine, riboflavine and pantothenic acid are essentially required for the growth.
• the growth is promoted by biotin and casamino acid.
• the strains which can be used according to the present invention include not only these three strains but also other strains of Pseudomonas sorbosoxidans inclusive of the mutants derived from these strains by irradiation with ultraviolet light or X-rays, or treatment with chemical mutagens such as N-methyl-N'-nitro-N-nitrosoguanidine (nitrosoguanidine), methylmethanesulfonate, nitrogen mustard and so on, as far as the strains are able to oxidize L-sorbose to 2-keto-L-gulonic acid.
• chemical mutagens such as N-methyl-N'-nitro-N-nitrosoguanidine (nitrosoguanidine), methylmethanesulfonate, nitrogen mustard and so on
• strain SB-15 which was derived from the strain 526-21 by treating with nitrosoguanidine.
• This mutant strain SB-15 exhibits the same taxonomical characteristics as the parent strain except that it shows an increased ability to produce 2-keto-L-gulonic acid from L-sorbose.
• the strain SB-15 was deposited at IFO on Apr. 23, 1987 as the accession number IFO 14606 and at FRI on May 1, 1987 as the accession number FERM BP-1356 under the Budapest Treaty.
• any of said strains can be cultivated with a medium containing L-sorbose or L-sorbose can be contacted with a processed material of said strains.
• processed material used in the present invention means washed cells obtained from culture broths of any of said strains, acetone dried cells, immobilized cells on supports such as polyacrylamide gel, ⁇ -carrageenin and the like.
• the starting material L-sorbose
• the starting material can be added all at once to a medium at the beginning of cultivation, or it may be added in several installments in the course of cultivation, or continuously to the culture medium.
• the concentration of L-sorbose based on the medium is 3 to 30% (w/v), preferably, 5 to 25% (w/v).
• L-sorbose As the method for contacting L-sorbose with said processed material, there can be employed, for example, a method wherein L-sorbose, 2-(N-morpholino)-ethanesulfonic acid (MES) buffer (0.5M, pH 6.5) and CaCO 3 are added to the processed material and the mixture is diluted with water, and shaken in a conical flask.
• MES 2-(N-morpholino)-ethanesulfonic acid
• CaCO 3 CaCO 3
• the concentration of L-sorbose is 0.1 to 10% (w/v), preferably 0.3 to 3% (w/v). and the amount of the processed material is 1 to 30 mg/ml, preferably 3 to 20 mg/ml, on the dried cell basis.
• the pH of the reaction solution is adjusted to 5.5 to 7.5 and the reaction temperature is about 20° to 40° C., the reaction time is about 1 to 100 hours.
• the medium used for cultivation of said strain can be liquid or solid so far as it contains nutrient sources which can be utilized by said strains. However, a liquid medium is preferred for mass production of the objective compound.
• carbon sources, nitrogen sources, inorganic salts, organic salts and trace nutrients which are used in conventional cultivation of microorganisms can be used.
• the starting material L-sorbose
• other auxiliary carbon sources such as glucose, glycerin, sucrose, lactose, maltose, molasses, and the like can also be used.
• the nitrogen sources there can be used inorganic and organic nitrogen-containing materials such as ammonium salts, corn steep liquor, peptone, meat extract, yeast extract, dry yeast, cottonseed meal, urea and the like.
• the inorganic salts include salts of potassium, sodium, calcium, magnesium, iron, manganese, cobalt, zinc, copper and phosphoric acid.
• biotin, thiamine, riboflavine, pantothenic acid and amino acids which are essential growth factors or promoters of said strains, or natural materials containing them.
• Stationary culture, shaking culture, or agitating culture with aeration can be employed as the means for cultivation.
• the so-called submerged culture is preferred.
• cultivation is preferably carried out at 25° to 35° C. and the pH of the medium is desirably about 5 to 9.
• 2-keto-L-gulonic acid is accumulated in the highest concentration.
• a suitable basic substance such as sodium hydroxide, potassium hydroxide, ammonia and the like to always maintain the optimal pH level for the microbial production of 2-keto-L-gulonic acid, or add a suitable buffer agent to maintain the optimal.
• the 2-keto-L-gulonic acid thus produced and accumulated in the culture broth of the reaction mixture can be harvested and purified by a per se known method utilizing its properties.
• 2-Keto-L-gulonic acid may be harvested in the form of the free acid, or isolated in the form of a salt of sodium, potassium, calcium, ammonium or the like.
• the culture broth is freed of cells, as required, by filtration, centrifugation or treatment with activated carbon and then the solution obtained is concentrated.
• the precipitated crystals are collected by filtration and further recrystallized to recover the objective compound. Further, precipitation, solvent extraction, chromatography or salting-out and other procedures may be applied in a suitable combination and/or in repetition.
• 2-keto-L-gulonic acid When 2-keto-L-gulonic acid is obtained in its free form, it can be converted to a salt of, for example, sodium, potassium, calcium, ammonium or the like by a conventional method. When it is obtained in the form of a salt, it can be converted into the free form or a different salt by a conventional method.
• the objective product obtained according to the present invention has been identified as 2-keto-L-gulonic acid by the determination of physicochemical properties such as elemental analysis, melting point, optical rotation, infrared absorption spectrum and the like.
• the quantitative determination of 2-keto-L-gulonic acid produced in the reaction mixture or the culture broth was performed by high performance liquid chromatography (mobile phase: diluted sulfuric acid to pH 2.2; flow rate: 0.5 ml/min.; detector: differential refractometer) using a sulfonated polystyrene gel column (manufactured by Shimadzu Seisakusho, Ltd., SCR-101H column, 7.9 mm ⁇ 30 cm). As the standard, crystals of sodium 2-keto-L-gulonate monohydrate were used. The detection of 2-keto-L-gulonic acid was conducted by thin layer chromatography.
• a 200 ml conical flask was charged with 20 ml of a seed culture medium containing 2.0% of glucose, 1.0% of polypeptone (Daigo Nutritive Chemicals, Japan) 0.5% of dried yeast and 2.0% of CaCO 3 and sterilized by autoclaving at 121° C. for 15 minutes.
• the flask was inoculated with a loopful of Pseudomonas sorbosoxidans 526-21 (IFO 14501; FERM P-8750; FERM BP-1334) grown at 28° C. for 2 days on a slant containing 2.0% glucose, 1% polypepton, 0.2% yeast extract, 0.5% NaCl and 1.5% agar, and incubated at 28° C. with shaking (200 rpm) for 2 days to obtain a seed culture broth.
• a sterilized 200 ml-conical flask was charged with 25 ml of a fermentation medium containing 1.0% polypepton, 0.2% casamino acid (Difco Lab. U.S.A.), 0.5% dried yeast, 0.5% (NH 4 ) 2 SO 4 , 0.05% Na 2 S 2 O 3 .5H 2 O, 0.03% KH 2 PO 4 , 0.05% MgSO 4 .7H 2 O, 0.1% FeSO 4 .7H 2 O, 0.0005% MnSO 4 .nH 2 O, 0.0005% thiamine hydrochloride and 6.0% CaCO 3 which were previously autoclaved, and 15.0% L-sorbose which was separately sterilized by filtration.
• a fermentation medium containing 1.0% polypepton, 0.2% casamino acid (Difco Lab. U.S.A.), 0.5% dried yeast, 0.5% (NH 4 ) 2 SO 4 , 0.05% Na 2 S 2 O 3 .5H 2 O, 0.03% KH 2 PO 4
• This conical flask was inoculated with 2 ml of the above-obtained seed culture broth and incubated at 28° C. with shaking for 3 days.
• An assay by the high performance liquid chromatography showed that the resulting fermentation broth contained 54.9 mg/ml of 2-keto-L-gulonic acid (conversion ratio: 34.0% based on used sugar).
• This fermentation broth 1,000 ml was centrifuged to remove the cellular and other sediments. The supernatant (about 980 ml) obtained was passed through an Amberlite IR 120 (Rhom & Haas Co., U.S.A., H-form, 500 ml) column which was then washed with about 300 ml of deionized water.
• a seed culture broth of Pseudomonas sorbosoxidans 526-22 (IFO 14502, FERM P-8751, FERM BP-1335) was prepared. Two ml of this seed culture broth was inoculated into a 200 ml-conical flask containing 25 ml of the same fermentation medium as that used in Example 1 except that the concentration of Na 2 S 2 O 3 .5H 2 O was changed to 0.1% and the flask was incubated with shaking at 30° C. for 5 days. In the resulting fermentation broth, there was contained 72.9 mg/ml of 2-keto-L-gulonic acid (conversion ratio: 45.1% based on used sugar).
• a 200 ml-conical flask was charged with 20 ml of a seed culture medium containing 2.0% glucose, 0.3% yeast extract, 0.3% CSL, 0.5% casein, and 2.0% CaCO 3 and sterilized by autoclaving at 121° C. for 15 minutes.
• the flask was inoculated with a loopful of Pseudomonas sorbosoxidans 526-42 (IFO 14503, FERM P-8752, FERM BP-1336) grown on the same slant as in Example 1 at 28° C. for 2 days, and incubated at 28° C. for 1 day to obtain a seed culture broth.
• a 200 ml-conical flask was charged with 20 ml of a medium containing 2.0% CSL, 0.05% Na 2 S 2 O 3 .5H 2 O, 0.1% FeSO 4 .7H 2 O, 0.3% (NH 4 ) 2 SO 4 , 0.0001% FMN, 0.00005% biotin and 9% CaCO 3 and sterilized by autoclaving.
• the flask was inoculated with 2 ml of the above-obtained seed culture broth and incubated with shaking at 30° C.
• the resulting fermented broth thus obtained contained 85.4 mg/ml of 2-keto-L-gulonic acid (conversion ratio: 50.3% based on used sugar; 86.0% based on consumed sugar).
• the fermented broth of Pseudomonas sorbosoxidans 526-21 (1,000 ml) as obtained in Example 1 was centrifuged and the resulting precipitate was suspended in a cold physiological saline solution (0.85%). The suspension was centrifuged at 1,000 rpm for 5 minutes to remove a precipitate mainly composed of CaCO 3 . The supernatant was further centrifuged at 9,000 rpm for 10 minutes to obtain washed cells. The cells were suspended in 35 ml of a cold physiological saline solution.
• the strain Pseudomonas sorbosoxidans 526-21 was grown on a slant at 30° C. for 3 days and the slant contained 2.5% D-sorbit, 1% peptone, 1% yeast extract, 0.2% CaCO 3 and 2% agar.
• a test tube charged 5 ml of PY medium (pH 7.0) containing 0.5% peptone, 0.5% yeast extract and 0.2% NaCl was inoculated with a loopful of cells from the slant and then incubated at 28° C. with shaking for 16 hours.
• a portion (2 ml) of the obtained culture broth was added to 1 ml of PY medium containing 1 mg nitrosoguanidine, and then incubated at 28° C. for 30 minutes.
• the treated cells were centrifuged at 5,000 rpm for 10 minutes, resuspended in 10 ml of fresh PY medium, and again centrifuged at 5,000 rpm for 10 minutes to remove the mutagen.
• Cells were suspended in 5 ml of PY medium and incubated at 28° C. with shaking for 3 hours.
• a loopful of cells grown on each of the slants was inoculated into a test tube poured with 3 ml of a medium (pH 6.6) composed of 10% L-sorbose, 2% corn steep liquor, 0.3% dried yeast, 0.02% Na 2 S 2 O 3 .5H 2 O, 0.1% FeSO 4 .7H 2 O, 0.3% (NH 4 ) 2 SO 4 , 0.4% peptone and 4% CaCO 3 and incubated at 30° C. with shaking for 5 days.
• a medium pH 6.6
• Each of the obtained culture broths was centrifuged at 12,000 rpm for 5 minutes, and the supernatant was diluted five times with 0.3N HCl, and again centrifuged at 12,000 rpm for 5 minutes.
• the mutant strain SB-15 shown in Example 5 was grown on a slant (pH 7.0) containing 2.5% D-sorbit, 1% peptone, 1% yeast extract, 0.2% CaCO 3 and 2% agar at 30° C. for 3 days.
• a loopful of the cells was inoculated into a sterile, 200 ml-conical flask which contained 20 ml of a medium (pH 6.8) containing 2% glucose, 1% peptone, 1% yeast extract and 2% CaCO 3 , and then incubated at 30° C. with shaking (200 rpm) for 2 days to obtain the first seed culture.
• a medium pH 6.8 containing 2% glucose, 1% peptone, 1% yeast extract and 2% CaCO 3
• the first seed culture (1.5 ml) was transplanted into a 200 ml-flask containing the above-mentioned medium and then incubated at 30° C. for 2 days to obtain the second seed culture.
• the second seed culture (2 ml) was transplanted into a sterile, 200 ml-conical flask poured with 25 ml of a fermentation medium containing 0.5% yeast extract, 2% corn steep liquor, 0.1% FeSO 4 .7H 2 O, 0.05% Na 2 S 2 O 3 .5H 2 O, 4% CaCO 3 and 10% L-sorbose (separately sterilized), and then incubated at 30° C. with shaking for 3 days.
• the obtained culture broth was found to contain 82.0 mg/ml of 2-keto-L-gulonic acid (conversion ratio: 76.1% based on used sugar) by subjecting high performance liquid chromatography, while the parent strain 526-21 incubated in the same manner was found to contain 47.1 mg/ml of 2-keto-L-gulonic acid.
• the strain SB-15 was incubated in the same manner as mentioned in Example 6 except for that concentrations of CaCO 3 and L-sorbose in the fermentation medium were 6 and 13%, respectively.
• the obtained culture broth was found to contain 84.0 mg/ml of 2-keto-L-gulonic acid (conversion ratio: 60.0% based on used sugar) by subjecting high performance liquid chromatography, while the parent strain 526-21 incubated in the same manner was found to contain 63.8 mg/ml of 2-keto-L-gulonic acid.

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• 1987
  • 1987-05-28 JP JP62133113A patent/JPH0795957B2/ja not_active Expired - Fee Related
  • 1987-06-04 US US07/057,979 patent/US4892823A/en not_active Expired - Lifetime

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